Tetravalent dengue vaccine

ABSTRACT

The invention provides a recombinant polypeptide comprising the EDIII domain of each of Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 linked to the N-terminal of HBsAg.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is filed under 35 U.S.C. § 371 as the U.S.national phase of International Application No. PCT/IB2015/056352, filedAug. 21, 2015, which designated the U.S. and claims the benefit ofpriority to India Patent Application No. 2478/DEL/2014 filed 1 Sep.2014, each of which is hereby incorporated in its entirety including alltables, figures and claims.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 26, 2017, isnamed ICG006US_SEQUENCE_LISTING_ST25_2665746.txt and is 17.5 kilobytesin size.

FIELD OF THE INVENTION

The present invention relates to a recombinant dengue subunit vaccineagainst all the four serotypes-DENV-1, DENV-2, DENV-3, DENV-4 of Dengue.The present invention also relates to a recombinant VLP-based denguequadrivalent vaccine candidate comprising tetravalent EDIII-T moleculeand surface antigen of Hepatitis B virus (HBsAg). The present inventionalso relates to a process for the production of recombinant VLP (viruslike particle)-based dengue quadrivalent vaccine candidate.

BACKGROUND OF THE INVENTION

Dengue disease caused by four antigenically distinct dengue viruses(DENVs) is a serious health concern in more than 150 countries of theworld and especially in highly endemic countries like India. Thisdisease has been on the rise since the last decade and has become aglobal public health threat because of lack of effective vaccine orantiviral therapies. Dengue disease is a global challenge for healthcaresystems particularly during outbreaks, and millions of dollars are spentevery year for vector control. An efficient and safe vaccine that iscost-effective could resolve the burden that dengue virus imposes onaffected countries. Despite intensive efforts over the past threedecades to develop a prophylactic vaccine for limiting the spread ofdisease, there is no licensed vaccine in the market as yet. Researchgroups/companies all over the world are undertaking efforts to developan effective tetravalent vaccine against all serotypes of Dengue virus.Earlier most of the vaccines developed were based on live, attenuated,chimeric viruses and some of them are currently under clinical trials.However, due to limitations such as viral interference, the researchfocus has transitioned towards subunit vaccines particularly using thedomain III of the envelope (E) protein of the dengue virus. Numerouspatents/publications exploiting this domain have also been reported.

Though a live flavivirus based dengue vaccine has entered phase IIIclinical trials, problems due to viral interference have been reported.Viral interference arises presumably because of differences inreplicative potential and immunogenicity of the four vaccine virusstrains.

Non-replicative subunit vaccines have the potential to overcome the riskof viral interference associated with live virus vaccines [Swaminathan,Khanna, N. (2009), Dengue: Recent advances in biology and current statusof translational research, Current Mol. Med. 9: 152-173]. Severalapproaches using recombinant DNA and protein based subunit vaccines arebeing explored. The majority of such recombinant subunit vaccines focuson the major envelope (E) protein. A number of evidences have furthershown that many of the vaccine properties of the E protein areassociated with domain III (EDIII).

DENV envelope domain III (EDIII) has been shown to be responsible forrecognition of the host cell receptor and generation of neutralizingantibodies [Swaminathan, Khanna, N. (2009). Dengue: Recent advances inbiology and current status of translational research, Current Mol. Med.9: 152-173; Guzman, M. G. Hermida, L., Bernardo, L., Ramirez, R.,Guillen, G. (2010). Domain II of the envelope protein as a denguevaccine target]. Moreover, EDIII has been reported to have only a verylow intrinsic potential for inducing cross-reactive anti-bodies[Simmons, M., Nelson, W. M., Wu, S. J., Hayes, C. G. (1998). Evaluationof the protective efficacy of a recombinant dengue envelope B domainfusion protein against dengue 2 virus infection in mice; Am. J Trop.Med. Hyg. 58: 655-662; Simmons, M, Murphy, G. S., Hayes, C. G. (2001).Short report: anti-body responses of mice immunized with a tetravalentdengue recombinant protein subunit vaccine, Am. J. Trop. Med. Hyg. 65:159-161]. These attributes make EDIII an excellent vaccine candidate.The efficacy of EDIII as a potential dengue vaccine antigen in the formof tetravalent protein has already been established by the inventors[Etemad, B., Batra, G., Raut, R., Dahiya, S., Khanam, S., Swaminathan,S., Khanna, N. (2008)].

Numerous patents/publications exploiting domain III of the envelope (E)protein of the dengue virus have been reported viz,

Suzarte, E., Gil, L., Valdés, I., Marcos, E., Lazo, L., Izquierdo, A., .. . & Hermida, L. (2015), International immunology, dxv011 discloses anovel tetravalent formulation combining the four aggregated domainIII-capsid proteins from dengue viruses induces a functional immuneresponse in mice and monkeys. This reference teaches a vaccine candidateagainst dengue virus based on two different viral regions, the domainIII of the envelope protein and the capsid protein, wherein thetetravalent formulation of DIIIC proteins was used. The novel chimericprotein from dengue-2 virus (domain III-capsid (DIIIC-2)), whenpresented as aggregates incorporating oligodeoxynucleotides, inducedantiviral and neutralizing antibodies, cellular immune response, andconferred significant protection to mice and monkeys. The remainingconstructs were already obtained and properly characterized. Based onthese evidences the present work was aimed at assessing the immuneresponse in mice of the chimeric proteins DIIIC of each serotype, asmonovalent and tetravalent formulations. The present inventorsdemonstrated the immunogenicity of each protein in terms of humoral andcell-mediated immunity, without antigen competition on the mixtureforming the formulation tetra DIIIC. Accordingly, significant protectionwas afforded as measured by the limited viral load in the mouseencephalitis model. The assessment of the tetravalent formulation innon-human primates was also conducted. In this animal model, it wasdemonstrated that the formulation induced neutralizing antibodies andmemory cell-mediated immune response with IFN-γ-secreting and cytotoxiccapacity, regardless the route of immunization used. The tetravalentformulation of DIIIC proteins constitutes a promising vaccine candidateagainst dengue virus.

Zuest, R., Valdes, I., Skibinski, D., Lin, Y., Toh, Y. X, Chan, K., . .. & Fink, K. (2015), Vaccine 33(12), 1474-1482 discloses theimmunogenicity of a tetravalent formulation of a recombinant fusionprotein consisting of E domain III and the capsid protein of dengueserotypes 1-4 (TetraDIIIC) to impart immunity against the dengue virus.E domain III is an epitope for efficient neutralizing antibodies whilethe capsid protein contains T cell epitopes. Besides combining B and Tcell epitopes, Tetra DIIIC is highly immunogenic due to its aggregateform and a two-component adjuvant. Following previous studies assessingthe monovalent DIIIC formulations, were addressed the quality andbreadth of the T cell- and antibody response of Tetra DIIIC in mice.Tetra DIIIC induced a Th1-type response against all four DENV serotypesand dengue-specific antibodies were predominantly IgG1 and IgG2a andneutralizing, while the induction of neutralizing antibodies wasdependent on IFN signaling. Importantly, the Th1 and IgG1/IgG2a profileof the DIIIC vaccine approach is similar to an efficient naturalanti-dengue response.

Izquierdo, A., Garcia, A., Lazo, L., Gil, L., Marcos, E., Alvarez, M., &Guzman, M. G. (2014), Archives of Virology, 159(10), 2597-2604 disclosesa tetravalent dengue vaccine containing a mix of domain III-P64k anddomain III-capsid proteins induces a protective response in mice. Thisreference teaches a vaccine candidate containing domain III of thedengue virus (type 1, 3 and 4) envelope protein fused to the P64kprotein from Neisseria meningitidis and domain III of dengue virus type2 (D2) was found to be immunogenic. Recombinant fusion proteinscontaining domain III of the dengue virus envelope protein fused to theP64k protein from Neisseria meningitidis and domain III of dengue virustype 2 (D2) fused to the capsid protein of this serotype wereimmunogenic and conferred protection in mice against lethal challenge inmice immunized with this tetravalent formulation were evaluated.

Live attenuated vaccines (LAVs), considered the most effective approachfor dengue, have belied this expectation. Recent data from an efficacytrial of the most advanced LAV candidate showed an overall efficacy of30%, with no efficacy for DENV-2. This necessitates serious explorationof alternate approaches to develop a dengue vaccine. VLP-based denguequadrivalent vaccine candidate, ‘DSV⁴’, is an HBsAg-based VLP displayingall the four EDIIIs corresponding to the four serotypes of DENVdeveloped.

Initially, as disclosed in Indian Patent No. 261749, a TetravalentDomain III protein (rTDIII), a single chimeric polypeptide comprisingdomain III of all four serotypes of Dengue virus, Dengue −1, 2, 3 and 4,linked with each other through penta-glycine linkers, wascodon-optimised for expression in E. coli.

An another Indian Patent application No. 1259/DEL/2007 discloses arecombinant envelop domain-III based tetravalent protein with andwithout secretory signal peptide eliciting protective immune responsesagainst each of the four serotypes of dengue virus, DEN-1, DEN-2, DEN-3and DEN-4, the said protein encoded by a polynucloeotide sequence codonoptimized for expression in eukaryotic expression system.

Ability of Hepatitis B surface antigen (HBsAg) to serve as a platformfor the presentation and display of foreign epitopes is illustrated wellby the success of malarial vaccine candidate RTS,S. In order to increasethe immunogenicity of EDIII-T, the inventors have explored whether HBsAgcould serve well for its display. Therefore, EDIII-T was cloned infusion with HBsAg and in a background of four expression cassettes ofHBsAg in P. pastoris vector (FIG. 1A). This design of DSV⁴ is similar tothat of RTS,S (Patent family: WO9310152 A1, MX9206574 A, EP0614465 A1,etc.), which displays malarial epitope on HBsAg VLPs.

The novelty of the present invention lies in the construct of EDIII-Twith HBsAg and in a background of four expression cassettes of HBsAg ina vector. Thus, this design of EDIII-T and HBsAg (termed as “DSV⁴”), canbe deemed novel. The inventiveness of the present invention lies in thefusion of the EDIII-T with the HBsAg to increase the immunogenicity ofthe expressed protein by serving as a platform for presentation and forthe co-expressed HBsAg protein to assemble into VLPs. A singlerecombinant tetravalent domain (EDIII-T) is cloned in fusion with HBsAgand co-expressed with HBsAg to form DSV⁴ Virus Like Particles (VLPs).The subunit tetravalent vaccine DSV⁴ generates DENV serotype specificneutralizing antibodies and is effective against each of the 4 serotypesof Dengue.

The present invention has/is expected to have the following advantagesover the existing proposed vaccines.

The Sanofi live attenuated vaccines currently under Phase-3 trialrequire three immunizations over an extended dosing schedule (0, 6 and12) of 12 months to elicit balanced neutralizing antibody responses toall 4 serotypes, while the Glaxo Smith Kline (in Phase 1 trials) isaiming at—2 doses 28 days apart—type of dosing schedule and Takeda(completed Phase 2 trials) with two doses separated by only threemonths, immunization regime. Thus, with the preliminary data, theimmunization schedule of the present vaccine would be shorter than theSanofi under-trial vaccine. Further, the present vaccine comprisesEDIII-T of all the 4 DENV serotypes and HBsAg as a single recombinantprotein whereas all the other vaccine candidates in trial includingSanofi's live attenuated virus vaccines are a mix of four candidatescorresponding to the four serotypes.

Further, the fusion of the recombinant EDIII-T with the HBsAg wouldresult not only in the formation of VLPs and co-expression of therecombinant immunogenic protein and the Hepatitis B surface antigen butcould provide protection/immunization against Hepatitis B along withdengue. This could lead to the development of a dual vaccine, providingsimultaneous immunization against all the serotypes of Dengue as well asHepatitis B.

OBJECTS OF THE INVENTION

An important object of the present invention is to provide a denguesubunit vaccine against all the four serotypes-DENV-1, DENV-2, DENV-3,and DENV-4.

Another object of the present invention is to provide a recombinantVLP-based dengue quadrivalent vaccine candidate, DSV4.

Yet another object of the present invention is to provide a recombinantVLP based dengue quadrivalent vaccine candidate, DSV4 which generatesDENV serotype specific neutralizing antibodies against each of the 4serotypes of Dengue, DENV-1, DENV-2, DENV-3 and DENV-4.

Still another object of the present invention is to provide a processfor the production of recombinant VLP-based dengue quadrivalent vaccinecandidate.

A further object of the invention is to provide an efficient and safevaccination approach against all the four serotypes of the Dengue virus.

SUMMARY OF THE INVENTION

In a first aspect the invention provides a recombinant polypeptidecomprising the EDIII domain of each of Dengue virus serotype DENV-1,DENV-2, DENV-3, and DENV-4 linked to the N-terminal of HBsAg.

In a further aspect the invention provides nucleic acid sequenceencoding a recombinant protein comprising the EDIII domain of Denguevirus serotype DENV-1, DENV-2, DENV-3, and DENV-4 linked to theN-terminal of HBsAg.

In a further aspect the invention provides a host cell transformed ortransfected with a nucleic acid of the invention, wherein the host cellexpresses HBsAg.

In a further aspect the invention provides a bio-nanoparticle comprisingthe recombinant polypeptide of the invention.

In a further aspect the invention provides a method of producing abio-nanoparticle comprising the recombinant polypeptide of theinvention, comprising culturing the host cell of the invention underappropriate conditions and recovering the expressed recombinant proteinor bio-nanoparticle.

In a further aspect the invention provides a vaccine comprising therecombinant polypeptide of the invention.

In a further aspect, the invention provides a vaccine comprising thebio-nanoparticle of the invention.

In a further aspect the invention provides a method of treating orpreventing Dengue virus, comprising administering to a subject therecombinant polypeptide of the invention, the bio-nanoparticle of theinvention or the vaccine of the invention.

In a further aspect the invention provides the recombinant polypeptideof the invention, the bio-nanoparticle of the invention or the vaccineof the invention for use in treating or preventing Dengue virus.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1: (A) Design of DSV⁴: EDIII-T consisting of four EDIIIscorresponding to the four DENVs linked through hexaglycyl linker wasgenetically fused with HBsAg (S) to encode for EDIII-T-HBsAg, which wascloned in vector carrying four expression cassettes of HBsAg. Therecombinant plasmid was linearized with Bg1 II and electroporated intoP. pastoris cells to obtain a clone co-expressing EDIII-T-HBsAg andHBsAg.

(B) Methanol induction: Uninduced (U) and induced (I) biomass ofselected clone was prepared by methanol induction and analysed forexpression by silver staining and western blotting. Silver stained gelshows expression of both EDIII-T-HBsAg (˜72 kDa) and HBsAg (˜25 kDa) ininduced sample. Western blot with EDIII specific mAb detectsEDIII-T-HBsAg, and with HBsAg specific mAb detects both EDIII-T-HBsAgand HBsAg.

(C) Purification of DSV⁴: Three preps of purified DSV⁴ from inducedbiomass.

FIG. 2: (A) Gel filtration chromatography of DSV⁴: DSV⁴ eluted in voidvolume when subjected to gel filtration chromatography. Analysis ofprotein in void volume by silver staining indicates presence of bothEDIII-T-HBsAg and HBsAg.

(B) CsCl ultracentrifugation: Co-migration of EDIII-T-HBsAg and HBsAg onultracentrifugation on CsCl column.

(C) Electron microscopic view of DSV⁴ VLPs: 25-35 nm sized DSV⁴ VLPs asobserved on negative staining under an electron microscope.

FIG. 3: (A) Source of EDIII sequence: Genotype of each DENV serotypefrom which corresponding EDIII aa sequence was derived.

(B) ELISA reactivity of DSV⁴ antiserum: Reactivity of pooled DSV⁴antiserum against HBsAg, EDIII-1, EDIII-2, EDIII-3 and EDIII-4. Purplecurve represents reactivity of un-immunized serum against DSV⁴.

(C) DSV⁴ generates balanced neutralizing titres: FACS-basedneutralization titres of DSV⁴ antiserum against specified genotype(s) ofeach serotype.

(D) Genotype neutralization breadth with pooled sera

FIG. 4: (a) the amino acid sequence of the envelope domain III of DENV-1(SEQ ID NO:1) and the encoding nucleic acid (SEQ ID NO:5);

(b) the amino acid sequence of the envelope domain III of DENV-2 (SEQ IDNO:2) and the encoding nucleic acid (SEQ ID NO:6);

(c) the amino acid sequence of the envelope domain III of DENV-3 (SEQ IDNO:3) and the encoding nucleic acid (SEQ ID NO:7);

(d) the amino acid sequence of the envelope domain III of DENV-4 (SEQ IDNO:4) and the encoding nucleic acid (SEQ ID NO:8);

(e) the amino acid sequence of the N-terminal of HBsAg (SEQ ID NO:9) andthe encoding nucleic acid (SEQ ID NO:10);

(f) the amino acid sequence of a recombinant polypeptide of theinvention comprising EDIII's from DENV-1, 2, 3 and 4 linked to theN-terminal of HBsAg (SEQ ID NO:11) and the encoding nucleic acid (SEQ IDNO:12), wherein the italicized nucleic acid and amino acid sequences arehexa-glycine linkers and the underlined nucleic acid and amino acidsequences are resulting from translation of KpnI restriction site. TheEDIII's are sequentially order from N-terminus to C-terminus DNV-1,DNV-3, DNV-4 and DNV-2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a dengue subunit vaccine against all thefour serotypes-DENV-1, DENV-2, DENV-3, and DENV-4 serotypes of Denguevirus. The subunit vaccine comprises a recombinant protein comprisingtetravalent EDIII-T and HBsAg. The present invention also relates to asubunit vaccine comprising VLP-based quadrivalent vaccine candidate forthe prevention of dengue disease against all the four serotypes of DENV.

In one aspect the invention provides a nucleic acid sequence encoding arecombinant protein comprising the EDIII domain of Dengue virus serotypeDENV-1, DENV-2, DENV-3, and DENV-4 linked in frame to the N-terminal ofHBsAg. The nucleotide sequences encoding each of the EDIII domains ofDengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 can be linkedwith the N-terminus of HBsAg in any sequential order.

Preferably, the nucleic acid sequence encodes EDIII domains of each ofDengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4, which have theamino acid sequences of SEQ ID NO's: 1, 2, 3 and 4 respectively.Preferably, the nucleic acid sequence encoding each of the EDIII domainsof Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 is SEQ IDNO's: 5, 6, 7 and 8 respectively.

Preferably, the nucleic acid sequence encodes a HBsAg having the aminoacid sequence of SEQ ID NO:9. Preferably, the nucleic acid sequenceencoding HBsAg is SEQ ID NO:10.

In one embodiment the nucleic acid sequence comprises each of thenucleotide sequences SEQ ID NO's: 5, 6, 7 and 8 linked in frame with theN-terminus of SEQ ID NO: 10. The nucleotide sequences SEQ ID NO's: 5, 6,7 and 8 can be linked with the N-terminus of SEQ ID NO: 10 in anysequential order.

Preferably, the nucleic acid sequence encodes a linker that links eachof the EDIII domains of Dengue virus serotype DENV-1, DENV-2, DENV-3,and DENV-4. Preferably, the nucleic acid encodes a flexible linker, mostpreferably a hexa-glycine linker. Preferably, the nucleic acid sequenceencodes a linker that links the EDIII domains of Dengue virus serotypeDENV-1, DENV-2, DENV-3, and DENV-4 to the N-terminal of HBsAg.Preferably, the nucleic acid encodes a flexible linker, most preferablya hexa-glycine linker.

Preferably, the nucleic acid sequence encodes a recombinant polypeptidehaving the amino acid sequence of SEQ ID NO: 11. Preferably, the nucleicacid sequence encoding the recombinant polypeptide is the nucleic acidsequence of SEQ ID NO:12.

In one embodiment the nucleic acid sequence is codon optimized forexpression in yeast, preferably for expression in P. pastoris. In oneembodiment the nucleic acid is an expression vector.

In one aspect the invention relates to a host cell transformed ortransfected with a nucleic acid of the invention, wherein the host cellexpresses HBsAg. In one embodiment the host cell is transformed ortransfected with a nucleic acid sequence that encodes HBsAg. Preferably,the host cell is transformed or transfected with 1, 2, 3, 4 or morenucleic acid sequences expressing HBsAg. In one embodiment the host cellis a yeast. Most preferably, the host cell is P. pastoris.

In one aspect the invention provides a recombinant polypeptidecomprising the EDIII domain of each of Dengue virus serotype DENV-1,DENV-2, DENV-3, and DENV-4 linked to the N-terminal of HBsAg. The aminoacid sequences encoding each of the EDIII domains of Dengue virusserotype DENV-1, DENV-2, DENV-3, and DENV-4 can be linked with theN-terminus of HBsAg in any sequential order. Preferably, EDIII domainsof Dengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 are fusedsequentially, N-terminal to C-terminal, in the sequence DENV-1, DENV-3,DENV-4 and DENV-2

Preferably, the amino acid sequence of the EDIII domains of each ofDengue virus serotype DENV-1, DENV-2, DENV-3, and DENV-4 is SEQ ID NO's:1, 2, 3 and 4 respectively. Preferably, the N-terminal of HBsAg has thepolypeptide sequence of SEQ ID NO:9. Preferably, the recombinantpolypeptide comprises each of the amino acid sequences SEQ ID NO's: 1,2, 3 and 4 linked to the N-terminus of SEQ ID NO:9. The amino acidsequences SEQ ID NO's: 1, 2, 3 and 4 can be linked with the N-terminusof SEQ ID NO: 9 in any sequential order. Preferably, the recombinantpolypeptide comprises the amino acid sequences SEQ ID NO's:1, 2, 3, and4 sequentially, N-terminal to C-terminal, in the sequence SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:2.

Preferably, the EDIII domains of each of Dengue virus serotype DENV-1,DENV-2, DENV-3, and DENV-4 are linked by a linker, preferably a flexiblelinker, most preferably a hexa-glycine linker. Preferably, the EDIIIdomains are linked to the N-terminal of HBsAg by a linker, preferably aflexible linker, most preferably a hexa-glycine linker.

In one aspect the recombinant polypeptide has the amino acid sequence ofSEQ ID NO: 11.

The transformed or transfected host cells of the invention synthesisesboth HBsAg and the recombinant polypeptide of the invention. Theinventors have shown that these two polypeptides spontaneouslyco-assemble into bio-nanoparticle. In one aspect the invention includesbio-nanoparticle comprising HBsAg and the recombinant polypeptide of theinvention of the invention.

In one aspect the invention provides a method of preparing a recombinantprotein or bio-nanoparticle comprising culturing the host cell of theinvention under appropriate conditions and recovering the expressedrecombinant protein or bio-nanoparticle.

In one aspect, the invention relates to a vaccine comprising therecombinant polypeptide or the bio-nanoparticles of the invention.Preferably, the vaccine comprises the recombinant polypeptide or thebio-nanoparticle of the invention in a pharmaceutically acceptablecarrier or suitable diluent.

In one aspect, the invention provides a method of treating or preventingDengue virus, comprising administering to a subject a recombinantprotein, a bio-nanoparticle or a vaccine of the invention. In oneembodiment the Dengue virus is serotype DENV-1, DENV-2, DENV-3, orDENV-4.

The tetravalent EDIII-based molecule, EDIII-T, was developed, which wasdesigned to contain all the four EDIIIs linked together through flexibleglycyl linkers as depicted in FIG. 1A. EDIII-T was expressed in Pichiapastoris, purified and was found to be immunogenic in mice. Theconstruction of the EDIII-T molecule is provided in the Indian PatentNo. 261749.

The ability of surface antigen of Hepatitis B virus (HBsAg) to serve asa platform for the presentation and display of foreign epitopes isillustrated well by the success of malarial vaccine candidate RTS,S inthe Patent Application No. WO 93/10152.

The present invention explores the possibilities whether HBsAg couldserve to increase the immunogenicity of EDIII-T. Therefore, EDIII-T wascloned in fusion with HBsAg and in a background of four expressioncassettes of HBsAg in P. pastoris vector as depicted in FIG. 1A. Thisdesign of EDIII-T and HBsAg is termed as “DSV⁴” and is similar to thatof RTS,S in the Patent application no. WO9310152 which displays malarialepitope on HBsAg VLPs.

The recombinant plasmid was electroporated in P. pastoris and thecolonies were screened for the co-expression of EDIII-T-HBsAg and HBsAgproteins by methanol induction of clones. One of the positive clonesco-expressing the two proteins as shown in FIG. 1B, was selected forfurther study. Induced biomass was lysed and the proteins associatedwith the membrane were extracted and subjected to diafiltration through300 kDa membrane. This step was designed to allow enrichment of largesized protein considering that the two co-expressed proteins assembleinto DSV⁴ VLPs. The retentate was purified through phenyl 600M resinwith high purity as depicted in FIG. 1C.

Ability of the co-expressed proteins to assemble into VLPs was evaluatedthrough gel filtration as shown in FIG. 2A, CsCl Ultra-centrifugation asshown in FIG. 2B and electron microscopy as shown in FIG. 2C. It wasobserved that both the protein components of DSV⁴ eluted together in thevoid volume during gel filtration as shown in FIG. 2A and co-migratedduring CsCl ultra-centrifugation as shown in FIG. 2B. On visualizationunder electron microscope, they were observed to assemble into 25-35 nmVLPs as shown in FIG. 2C.

The conformational integrity of EDIII of all four DENVs in DSV⁴ VLPs wasevaluated through recognition of critical EDIII epitopes by wellcharacterized mAbs in sandwich ELISA format.

Capability of these VLPs to mount a strong immune response against thefour DENV serotypes was evaluated by immunization in BALB/c mice asshown in FIG. 3. Purified DSV⁴ VLPs were immunized (20 μg/500 μg Al asalhydrogel/100 μl in PBS) in a group of six BALB/c miceintraperitoneally on days 0, 30 and 90. Terminal bleed was taken on day100 and analysed for response against DSV⁴ by ELISA. Sera from positiveresponders were pooled and characterized for the presence of antibodiesagainst all its five components namely EDIII-1, EDIII-2, EDIII-3,EDIII-4 and HBsAg (FIG. 3B). It was observed that a strong immuneresponse was generated against all of them. It was essential todetermine whether anti-dengue response was capable of neutralizing thefour DENVs. Therefore, the pooled serum was evaluated for itsneutralization capacity through FACS-based assay and it was observedthat DSV⁴-antiserum was indeed capable of neutralizing all four DENVs(FIG. 3C). FIG. 3A illustrates the design of DSV⁴ and the strain fromwhich corresponding EDIII aa sequences were acquired. FIG. 3Cillustrates the neutralization titre of DSV⁴-antiserum against the fourDENVs (of specified strain) and strains of two additional genotypes ofDENV-3. It is evident that DENV-2, -3 and -4 genotypes used inneutralization assay varied from the genotypes from which EDIII sequencewas acquired, and it did not adversely affect the neutralizationcapability of DSV⁴ antiserum, indicating the high strength of thegenerated immune response. Moreover, the overall response against thevarious genotypes also appeared to be balanced. highlighting thecandidacy of DSV⁴ as a potential dengue vaccine as depicted in FIG. 3D.DSV4 appeared to be efficacious to comparable extent with variousadjuvants evaluated (FIG. 3D)

The present invention is described with reference to the followingexamples, which are included merely to illustrate and demonstrate theinvention. These specific examples should not be construed to limit thescope of the invention in any way.

Example 1: Construction of Recombinant VLP-Based Dengue QuadrivalentVaccine Candidate

EDIII-T was cloned in fusion with HBsAg and in a background of fourexpression cassettes of HBsAg in P. pastoris vector as depicted in FIG.1A. This design of EDIII-T and HBsAg is termed as “DSV⁴” The recombinantplasmid was electroporated in P. pastoris and the colonies were screenedfor the co-expression of EDIII-T-HBsAg and HBsAg proteins by methanolinduction of clones.

Example 2: Characterization of Dengue Quadrivalent VaccineCandidate-DSV⁴

One of the positive clones co-expressing the two proteins as shown inFIG. 1B, was selected for further study. Induced biomass was lysed andthe proteins associated with the membrane were extracted and subjectedto diafiltration through 300 kDa membrane. This step was designed toallow enrichment of large sized protein considering that the twoco-expressed proteins assemble into DSV⁴ VLPs. The retentate waspurified through phenyl 600M resin with high purity as depicted in FIG.1C.

Example 3: Identification and Characterization of VLPs

Ability of the co-expressed proteins to assemble into VLPs was evaluatedthrough gel filtration as shown in FIG. 2A, CsCl Ultra-centrifugation asshown in FIG. 2B and electron microscopy as shown in FIG. 2C. It wasobserved that both the protein components of DSV⁴ eluted together in thevoid volume during gel filtration as shown in FIG. 2A and co-migratedduring CsCl ultra-centrifugation as shown in FIG. 2B. On visualizationunder electron microscope, they were observed to assemble into 25-35 nmVLPs as shown in FIG. 2C.

Example 4: Evaluation of Conformational Integrity of All Four DENVs inDSV⁴ VLPs by mAbs

The conformational integrity of EDIII of all four DENVs in DSV⁴ VLPs wasevaluated through recognition of critical EDIII epitopes by wellcharacterized mAbs in sandwich ELISA format. Dengue specific mAbs werecoated in microtiter wells and DSV⁴ VLPs were added. Bound VLPs wererevealed through peroxidase labelled anti-HBsAg Hepnostika. Most ofthese mAbs were against A-strand and lateral ridge region of EDIII,which are believed to be essential in generating a strong neutralizingimmune response. ELISA reactivity of DSV⁴ by 21 dengue mAbs (EDIII andnon-EDIII specific mAbs) is illustrated in Table 1 and the resultsindicate that EDIII epitopes of all four DENVs are intact in DSV⁴ VLPs.

TABLE 1 List of EDIII-specific mAbs, regions recognized by them andtheir reactivity with ‘DSV⁴’ VLPs (in terms of ELISA OD) Anti-EDIIIStrongly Region ELISA mAbs neutralizes specificity OD E103 DENV-1 L.R-BCloop 3.96 3H5 DENV-2 A.S and BC loop (L.R) 0.77 70 A.S 0.48 106 A.S 0.51104 C strand/CC′ loop 0.52 8A1 DENV-3 LR-N terminus of 1.30 A.S, FG loopE51 236 L.R 1.84 E51 202 L.R 3.90 E88 DENV-4 BC and DE loop (L.R) 0.53E76 DENV-4, -2 N terminal A.S, CC′ 0.51 loop, B, D and G strands E106DENV-1, -4 A.S and L.R 3.60 E113 DENV-1, -2, -4 L.R 1.20 h-2J20 DENV-1,-3 2.50 E61 DENV-1, -2, A.S and G strand 0.71 E77 -3, -4 A.S, BC loopand G 0.81 strand Non-EDIII Ab Region ELISA mAbs type specificity ODh-2K2 Complex prM 0.06 4G2 Fusion Loop 0.05 h-1M7 0.06 h-DVC23.13 0.053H4 0.05 h-1N5 0.05

Example 5: Immunization of Mice by Purified DSV⁴ VLPs

Capability of these VLPs to mount a strong immune response against thefour DENV serotypes was evaluated by immunization in BALB/c mice asshown in FIG. 3. Purified DSV⁴ VLPs were immunized (20 μg/500 μg Al asalhydrogel/100 μl in PBS) in a group of six BALB/c miceintraperitoneally on days 0, 30 and 90. Terminal bleed was taken on day100 and analysed for response against DSV⁴ by ELISA. Sera from positiveresponders were pooled and characterized for the presence of antibodiesagainst all its five components namely EDIII-1, EDIII-2, EDIII-3,EDIII-4 and HBsAg as depicted in FIG. 3B and further Genotypeneutralization breadth of DSV⁴ antisera generated with various adjuvantswas determined as depicted in FIG. 3D

Immune Response

It was observed that a strong immune response was generated against allof the five components namely EDIII-1, EDIII-2, EDIII-3, EDIII-4 andHBsAg. It was essential to determine whether anti-dengue response wascapable of neutralizing the four DENVs. Therefore, the pooled serum wasevaluated for its neutralization capacity through FACS-based assay andit was observed that DSV⁴-antiserum was indeed capable of neutralizingall four DENVs as shown in FIG. 3C which illustrates the neutralizationtitre of DSV⁴-antiserum against the four WHO reference strains DENVs andstrains of two additional genotypes of DENV-3. It is evident thatDENV-2, -3 and -4 genotypes used in neutralization assay varied from thegenotypes from which EDIII sequence was acquired, and it did notadversely affect the neutralization capability of DSV⁴ antiserum,indicating the high strength of the generated immune response. Moreover,the overall response against the various serotypes also appeared to bebalanced, highlighting the candidacy of DSV⁴ as a potential denguevaccine. Table 2 below illustrates FNT post depletion on EDIII-3-MBPeliciting serotype specific neutralizing Abs.

TABLE 2 FNT post depletion on EDIII-3-MBP elicits serotype specificneutralizing Abs Sera depleted FNT₅₀-Vero on DENV-1 DENV-2 DENV-3 DENV-4Sera 1: MBP 714 678 2259 415 Depletion of EDIII-3- 1276 581 288 449DENV-3 Abs MBP Sera 2: MBP nd 775 1022 nd Depletion of EDIII-2- nd 78975 nd DENV-2 Abs MBP

The invention also includes the following specific aspects:

Aspect 1. A recombinant VLP-based dengue quadrivalent vaccine candidatecomprising a tetravalent EDIII-T molecule and the surface antigen ofHepatitis B virus (HBsAg).

Aspect 2. A recombinant VLP-based dengue quadrivalent vaccine candidatedesignated DSV⁴.

Aspect 3. The recombinant VLP-based dengue quadrivalent vaccinecandidate, DSV⁴, wherein the tetravalent EDIII molecule comprises EDIIIof DENV-1, DENV-2, DENV-3, and DENV-4.

Aspect 4. The recombinant VLP based dengue quadrivalent vaccinecandidate as recited in aspect 2 of the invention, wherein DSV⁴generates DENV serotype specific neutralizing antibodies against DENV-1,DENV-2, DENV-3 and DENV-4.

Aspect 5. A process for the production of recombinant VLP-based denguequadrivalent vaccine candidate as claimed in claim 1 comprises the stepsof:

i) cloning of EDIII-T in fusion with HBsAg in a recombinant constructcarrying four expression cassettes of HBsAg;

ii) electroporation of recombinant plasmid into Pichia pastoris cells toobtain a clone co-expressing EDIII-T-HBsAg and HBsAg;

iii) screening for the co-expression of EDIII-T-HBsAg and HBsAgproteins;

iv) analyzing the expression of EDIII-T-HBsAg and HBsAg proteins bysilver staining and western blotting;

v) lysis of the induced biomass;

vi) extraction of the proteins associated with the membrane andsubjected to diafiltration;

vii) purification of DSV⁴

Aspect 6. The process as recited in aspect 5, wherein the screening instep (iii) is done by methanol induction of clones.

Aspect 7. A dengue subunit vaccine comprising a recombinant VLP-baseddengue quadrivalent vaccine candidate as recited in Aspect 1.

Aspect 8. The dengue subunit vaccine as recited in Aspect 7, whereinsaid vaccine is active against DENV-1, DENV-2, DENV-3, and DENV-4serotypes of Dengue virus.

Aspect 9. The dengue subunit vaccine as recited in aspect 8 wherein saidvaccine can be administered intraperitoneally or intramuscularly.

Aspect 10. A recombinant VLP-based dengue quadrivalent vaccine candidatefor use as a dengue subunit vaccine candidate comprising of tetravalentEDIII-T molecule and the surface antigen of Hepatitis B virus (HBsAg).

The invention claimed is:
 1. A virus like particle (VLP) comprising: a) a recombinant polypeptide comprising an EDIII domain of each of Dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4, linked to N-terminal of an HBsAg polypeptide, as depicted below: EDIII-1-EDIII-3-EDIII-4-EDIII-2-HBsAg; wherein the dotted line is a linker; and wherein the amino acid sequence of the EDIII domain of each of Dengue virus serotypes DENV-1, DENV-2, DENV-3 and DENV-4 is SEQ ID NO's 1, 2, 3 and respectively; and b) 4 units of an HBsAg polypeptide.
 2. A nucleic acid sequence encoding a recombinant polypeptide of claim
 1. 3. A virus like particle (VLP) of claim 1, wherein the recombinant polypeptide has an amino acid sequence according to SEQ ID NO
 11. 